Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

Transduction of endothelial cells (EC) with a vector that expresses apolipoprotein A-I (APOAI) reduces atherosclerosis in arteries of fat-fed rabbits. However, the effects on atherosclerosis are partial and might be enhanced if APOAI expression could be increased. With a goal of developing an expression cassette that generates higher levels of APOAI mRNA in EC, we tested 4 strategies, largely in vitro: addition of 2 types of enhancers, addition of computationally identified EC-specific cis-regulatory modules (CRM), and insertion of the rabbit APOAI gene at the transcription start site (TSS) of sequences cloned from genes that are highly expressed in cultured EC. Addition of a shear stress-responsive enhancer did not increase APOAI expression. Addition of 2 copies of a Mef2c enhancer increased APOAI expression from a moderately active promoter/enhancer but decreased APOAI expression from a highly active promoter/enhancer. Of the 11 CRMs, 3 increased APOAI expression from a moderately active promoter (2–7-fold; P < 0.05); none increased expression from a highly active promoter/enhancer. Insertion of the APOAI gene into the TSS of highly expressed EC genes did not increase expression above levels obtained with a moderately active promoter/enhancer. New strategies are needed to further increase APOAI transgene expression in EC.

To construct additional expression cassettes in which DNA sequences were tested for their ability to increase APOAI expression above levels obtained with the 4XETE-gApoAI-oPRE cassette, we first used site-directed mutagenesis to insert MluI and BglII restriction sites upstream of the 4XETE sequence in pBShuttle-4XETE-gApoAI-oPRE, generating pBShuttle-MB-4XETE-gApoAI-oPRE ( Supplementary Fig. S1). We then constructed an oligomer containing a 44-bp murine Mef2c enhancer, 8 flanked by MluI and BglII restriction sites on one end and a BamHI restriction site (Integrated DNA Technologies, Coralville, IA) on the other end.
We used a similar approach to construct expression cassettes in which single copies of endothelial cell (EC) cis-regulatory modules (CRM; identified as described below) were inserted upstream of the 4XETE and 1XETE sequences in pBshuttle-MB-4XETE-gApoAI-oPRE and pBshuttle-MB-1XETE-gApoAI-oPRE. Essentially, we used PCR and human DNA from 293 Cre cells 6 as a template to generate amplicons containing each of 11 CRM (all with introduced 5´ MluI sites and 3´ BglII sites), we digested the amplicons with MluI and BglII, and ligated the products into the MluI/BglII-digested plasmids. These expression cassettes were termed CRM(1-11)-1XETE-gApoAI-oPRE and CRM(1-11)-4XETE-gApoAI-oPRE ( Supplementary Fig.   S1).
We also constructed 4 expression cassettes in which APOAI expression was driven by large (2 035 -8 880 bp) genomic segments of each of 4 genes that are highly and relatively specifically expressed in EC: VWF, THBS1, EFEMP1, and CDH5. To identify these 4 genes, we began by using publicly available search engines and PubMed, along with search terms that included "endothelium genes," "endothelium-specific genes," and "endothelial cell specific." These searches identified 30 candidate genes (Supplemental Table 1). We consulted the relevant publications and confirmed that each publication reported expression of these genes in EC. We then consulted the Gene Expression Atlas (http://www.ebi.ac.uk/gxa/home), which includes quantitative transcription data generated from the ENCODE project. 10 For each of the 30 genes, we interrogated ENCODE-derived data in the Gene Expression Atlas using: endothelial cell-derived cell line, the gene name, ENCODE -long polyA RNA, and whole cell.
The output of this interrogation yielded a table that reports relative expression levels of the 30 genes in 18 cell lines, including cultured human umbilical vein EC (HUVEC; Supplementary Table S1). EFEMP1 and THBS1 were by far the most highly expressed genes in HUVEC and had relatively EC-specific expression. VWF and CDH5 were the next most highly expressed genes in HUVEC and were expressed only in HUVEC.
To determine which genomic sequences to incorporate in the 4 expression cassettes, we consulted papers that identified cis-acting positive regulators of transcription located near the promoters of the 4 genes. 11- 21 We were also careful to include the genomic regions in which the CRM were located (see below). Into these genomic sequences, we ligated a cassette that contains elements of the rabbit APOAI gene (beginning with the start codon, and including all 3 coding exons, both introns, and 51 bp of 3´ untranslated region), as well as the oPRE and SV40 polyadenylation signal. This APOAI cassette was inserted at the translational start sites of each of these 4 genomic sequences ( Supplementary Fig. S2). The VWF genomic sequence extends from 843 bp upstream of the transcription start site to the translation start site, including the promoter, first exon, and first intron (total 2 322 bp). We used 2 segments of the EFEMP1 gene, with one segment placed upstream and one segment downstream of the APOAI gene.
The upstream sequence extends from 243 base pairs upstream of the EFEMP1 transcription start site to immediately upstream of the EFEMP1 start codon, including the promoter, first exon, first intron, and part of the second exon (total 1 942 bp). The 3´ segment begins immediately downstream of the EFEMP1 start codon, extends for 5 676 base pairs downstream of the EFEMP1 translation start site (including the 2nd, 3rd, and 4th exons, the 2nd and 3rd introns, and part of the 4th intron). We used 2 separate segments of the EFEMP1 gene because CRM were identified both 5´ and 3´ of the EFEMP1 translation start site, and because leaving exon 2 (containing the EFEMP1 transcription start site) intact would potentially result in transcription and translation of a chimeric mRNA that included both EFEMP1 and APOAI sequences. The THBS1 genomic sequence extends from 1 270 bp upstream of the transcription start site to the translation start site, including the promoter, first exon, first intron, and part of the second exon (total 2 035 bp). The CDH5 genomic sequence includes 2 segments. The first segment extends from 6 721 bp upstream of the transcription start site through part of the first intron (total 8 821 bp). The second segment includes a sequence from the 3´ end of the first intron, including the splice acceptor site, and part of the second exon (total 59 bp). A plasmid containing the second segment for CDH5 and part of the 3´ end of the first segment was constructed by Integrated DNA Technologies.
Supplementary Figure S2. Genomic knock-in expression cassettes. An engineered rabbit APOAI gene was inserted at the translation start sites (ATG) of genomic sequences cloned from the human VWF, EFEMP1, THBS1, and CDH5 loci. The engineered APOAI gene includes the 3 APOAI coding exons (yellow boxes), beginning with the ATG in native exon 2, 2 APOAI introns (grey bars), a segment of APOAI 3´ untranslated region (3U), the optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), and an SV40 poly A signal (pA). Cloned segments of human genomic DNA in the cassettes include: sequences 5´ to the transcription start sites (blue boxes), the transcription start sites (+1), exons of VWF, EFEMP1, THBS1, and CDH5 (yellow boxes numbered 1-4), introns (i), and the translation start sites (ATG). Green bars indicate positions of cis-regulatory modules identified in silico (see Materials and Methods). All cassettes were constructed in the pBshuttle plasmid vector (v). Junctions of the expression cassettes and pBshuttle are indicated (* and **). Drawing is not to scale.